The early growth response (Egr) family of transcription factors is composed of four members Egr1-4, which play roles in various cell behaviors including proliferation, apoptosis, and differentiation in a cell type and stimulus specific manner. Roles for Egr1-3 are especially well documented in the nervous system: Egr 1 and 3 regulate expression of genes involved in synaptic plasticity and long term potentiation, Egr2 contributes to hindbrain development and peripheral nerve myelination, and Egr 1 and 2 also contribute to the transcriptional network that drives neuronal differentiation in PC12 cells. However, the mechanisms that regulate Egr activity are not fully understood. One mechanism of Egr regulation involves interactions with coregulators NGFI-A binding protein 1 and 2 (Nab1 and Nab2). Most studies demonstrate that Nab1 and 2 repress Egr transactivation when bound; however, one report indicates Nabs can enhance Egr transactivation in a target gene-specific manner. As such, the molecular mechanism controlling the effect of Nabs on the activity of Egr1-3 remain unclear. Here, we provide evidence that Nab2 is rapidly phosphorylated in PC12 cells following treatment with nerve growth factor (NGF) based on an upward mobility shift in Western blot that is reversed by phosphatase treatment. The mobility shift was inhibited by the mitogen-activated protein kinase kinase (MEK) inhibitor (UO126), indicating Nab2 phosphorylation is mediated at least in part by extracellular-regulated kinase (ERK). Nine sites on Nab2 that match the consensus sequence for ERK phosphorylation were identified and site-directed mutagenesis was conducted to generate alanine mutants for each. Unexpectedly, exogenous Nab2 was constitutively phosphorylated independent of NGF treatment; nevertheless, the Nab2-S126A, -S129A, -S138A, T157A, and -T401A mutants exhibited downward mobility shifts, indicating they are likely phosphorylation sites. Mammalian two-hybrid assays were then conducted to detect Nab2-Egr1 interactions and to evaluate whether the mutation of each of the phosphorylation sites affect their interactions. Experiments thus far have tested the effect of three Nab2 phosphorylation site mutants (S126A, T157A, and S401A), none of which affect Nab2 interactions with Egr1.
Kenneth Adams (Thesis Advisor)
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Original document was submitted as an Honors Program requirement. Copyright is held by the author.
Abt, Kimberly. (2018). Characterizing Nab2 Phosphorylation and its Role in Regulating Egr Transcription Factors. In BSU Honors Program Theses and Projects. Item 303. Available at: http://vc.bridgew.edu/honors_proj/303
Copyright © 2018 Kimberly Abt